Project Summary The action potential (AP) is a command signal that sharply controls the activity of voltage-gated Ca2+ channels (Cavs) and neurotransmission. The AP waveform has traditionally been considered to be a uniform, binary signal as it propagates across an axonal arbor, however recent work has suggested there is a surprising amount of variability in the width of the waveform arising from a heterogeneous distribution of voltage-gated Na+ and K+ channels across synapses. In the hippocampus, neurons burst in high frequency trains that undergo waveform broadening though it is unclear what the ramifications of this broadening are. A fundamental gap exists in understanding how variations in the AP waveform mechanistically affect neurotransmission as an experimental approach is required with subcellular resolution that can integrate at the microsecond time scale. The development of optogenetics has provided opportunities for manipulating and imaging activity within the small en passant synapses of the central nervous system such as in the hippocampus. These experiments will provide the first optical measurements of presynaptic APs, Ca2+ influx and exocytosis in single hippocampal neurons. Modulation of the AP waveform is achieved pharmacologically by inhibiting a family of voltage-gated K+ channels which results in a predictable broadening of the AP. An unexpected phenotype occurs with this treatment in excitatory neurons: a dramatic increase in exocytosis corresponds with a minimal increase in Ca2+, suggesting an uncoupling of the clearly defined Ca2+ and vesicle fusion relationship and an enhancement of synaptic efficacy. The central hypotheses of this proposal are that a broadened AP waveform alters the Ca2+ contribution of a specific Cav isoform, changes the radius of Ca2+ microdomains and/or differentially activates vesicle fusion machinery. The overall objective of this proposal is to investigate the mechanisms behind the transduction of an electrical action potential signal to the chemical release of neurotransmitter. The long-term goals are to determine how heterogeneity of the AP waveform informs synaptic strength and plasticity. Specific Aim 1 will determine how waveform broadening modulates presynaptic Cav isoform contribution using selective toxins to isolate specific isoforms with and without a broadened AP. Specific Aim 2 will determine how AP shape alters Ca2+ microdomains and vesicle exocytosis. Using optogenetics, gene silencing and pharmacology these experiments will demonstrate if AP broadening influences the radius of microdomains or differentially activates the protein Ca2+ sensors that mediate exocytosis. Given the complexity of this system as well as the essential nature of electrical signaling in excitable cells it is unsurprising that mutations in the voltage- gated channels which control the shape of the AP are implicated in several diseases including epilepsy, ataxia and arrhythmias. Due to the role the AP plays as an information carrier by modulating intracellular Ca2+ and subsequently neurotransmission, there is a critical need for a better understanding of this level of neural regulation.